There is increasing evidence that cytokines, in general, and tumor necrosis factor (TNF), in particular, may play a key role in the pathogenesis of Crohn's disease (CD), one of the known idiopathic inflammatory bowel diseases (IBD)s. The most compelling evidence supporting the role of TNF in CD comes from recent studies using single dose infusions of anti-TNF monoclonal antibody in patients active, therapy-refractory disease. In the majority of treated patients, TNF blockade resulted in decreased disease activity and sustained remission. Increasing evidence suggests that these effects are immunologically mediated. However, the precise mechanism(s) of TNF's actions in CD and the relationship between TNF and immune effector cells remains unclear. Our preliminary findings provide direct evidence that a specific deregulation of TNF synthesis in mice, i.e. targeted deletion of AU-rich elements (deltaARE) in the 3' UTR region of the TNF gene, results in systemic TNF over-expression and triggers a CD-like intestinal phenotype that closely resembles the human disease. Therefore, the central hypothesis of this proposal is that TNF play a crucial pathogenic role in CD by affecting primary immunoregulatory mechanisms, which result in the gut inflammatory changes characteristic of this disease. In this context, anti-TNF treatment may not directly target the effector functions of TNF but may, rather, interfere with these key immunological mechanisms. This hypothesis will be tested in TNF/deltaARE by investigating the following 4 specific aims: 1) Characterize the features of chronic intestinal inflammation. The clinical phenotype, histological features, as well as the specific cytokine profiles will be investigated in our TNF/deltaARE colony at UVA. 2) Define the role of the immune response in the development of intestinal. A series of cross-breeding studies, T -cell adoptive transfer as well as bone marrow chimera experiments will be performed to precisely define the role of T-cell subsets, B cells and other immunocytes in mediating the observed CD-like pathology. 3) Examine the role of the two TNF receptors in mediating intestinal inflammation. The expression of TNF receptor isoforms in intestinal tissues as well as isolated mucosal cells will be studied by multiple techniques. In addition, the deltaARE mutation will be introduced into TNFRI- or TNFRII-deficient mice to assess the relative contribution of the two TNF receptors to TNF-mediated gut inflammation. Finally, we will study the effects of gut-specific reactivation of the TNFRI and/or TNFRII in double mutant TNF/deltaARE/TNFR-/- mice. 4) Determine whether gut-specific TNF over-expression is sufficient to induce chronic intestinal inflammation. Using an intestinal-specific promoter and Cre-loxP technology, we will compare the effects of systemic versus local TNF over-expression on the development of chronic intestinal inflammation in TNF/deltaARE mice. The ultimate goal of the present research proposal is to define the precise mechanism(s) of TNF-induced CD in order to develop specific treatment modalities aimed at modifying the natural course of this devastating disease.